The tobacco alkaloids: nicotine, nornicotine, cotinine, anabasine, their protonated forms, and salts were analyzed by means of Raman spectroscopy supported by the density functional theory/B3LYP/aug-cc-pVDZ calculations. The analyses were per- formed based on Raman marker bands of neutral, monoprotonated, and diprotonated forms of tobacco alkaloids because in different surroundings various forms have been suggested to either dominate or to coexist. The form and distribution of nicotine directly in a plant and in phytopharmaceutical products were investigated by in situ Raman mapping. For the fi rst time, the Raman optical activity spectrum of (

)-nicotine in aqueous solution was measured and interpreted by means of the density functional theory calculations. The study provides a clear evidence that Raman spectroscopy techniques are pow- erful in ef fi cient quality control and forensic and bioanalytical analyses of tobacco alkaloids.